The invention provides compositions and methods for the detection of targets in a sample, including an in situ hybridization (ISH) sample. Probes and detectable labels may be provided in multiple layers in order to increase the flexibility of a detection system, and to enhance the signal from a target. The compositions and methods are compatible with a variety of detection systems, and with many different types of targets, probes, and detectable labels.
Detection of a target in a sample may ordinarily be achieved by contacting the target with a probe that specifically recognizes it. The probe may be linked, either directly or indirectly to a detectable label, such as a fluorophore or radioactive tag, which provides a signal representing the target.
Some detection systems also provide ways of enhancing the signal from the target. For example, the label's signal may be enhanced by increasing the number of detectable labels used to detect each target, or by instrumentation that may amplify the signal. Alternatively, if the target is an antigen, a multiple-antibody system may amplify the detection signal. For instance, the target may first be bound by a primary antibody, which, in turn, is capable of binding many secondary antibodies or even tertiary antibodies, which, in their turn, bind to the probe. This method, thus, increases the number of probes that recognize each antigen target by adding extra layers of molecular interactions between the probe and target.
The secondary antibody technique is widely used, but may be limited due to, for example i) the availability of the secondary antibodies; ii) unwanted cross reactivity between closely related species, e.g. rat, mouse; iii) the size of antibodies reduces the penetration of the reagents. Furthermore, conjugation of antibodies to other antibodies, enzymes, color labels, etc. is somewhat unique for every antibody due to biological variations. Fab or Fab2 fragments of IgG have been used to overcome the size and non-specific binding problems, but such secondary antibody based visualization systems still remain limited to staining of one, two or three different targets. Amplification of the signal from individual targets is both laborious and complex due to the above-mentioned technical limitations. Further, currently used antibody-based amplification methods may only be of practical use with certain types of targets and detectable labels. In contrast, the instant invention uses the flexibility of nucleic acid hybridization to provide a general set of compositions and methods that may be used to detect one or many targets in a sample and amplify their signals.
The instant methods and compositions may also provide for increased flexibility as compared to, for example, capture assays or sandwich assays, such as those described in U.S. Pat. No. 4,868,105, for example, that rely on one type of binding interaction such as a hapten-protein or primary antibody-secondary antibody interaction. The ability to design a variety of nucleic acid analog hybridization pairs, for instance, may dramatically increase the ways in which the probe and detectable label may interact.
In some embodiments of the invention, compositions and methods may separate the probe and the detectable label such that they may be comprised on different units within a detection system. See, for example, FIG. 1a and 1c. The units may then interact through specific hybridization of nucleic acid analog segments. The units may be designed such that a given probe may interact with a variety of different detectable labels, depending upon the needs of the assay. The units may also be designed to include multiple interacting nucleic acid analog segments, either to increase the affinity between the units, or to amplify a signal. Further, adaptor units may be included that provide, for example, one or more additional molecular layers between the probe and the detectable label. See FIG. 1b. In some embodiments, the adaptor units may allow for even greater mixing and matching of probes and labels as well as additional amplification of a signal. The components of the instant compositions may also be designed to be of similar chemical compositions so that they may be prepared simply using standardized conjugation schemes.
Due to the relative ease with which nucleic acid hybridization schemes may be planned, the methods and compositions of the invention may also be used to detect more than one target within a sample. For instance, multiple targets in a sample are not always expressed in equal amounts. Thus, there may be a differential need for amplification. The instant invention is also useful in normalizing the detection of two or more targets in a system.
The system may also be designed such that one unit specifically hybridizes to more than one other unit. For instance, the various units of the invention may be designed such that one unit hybridizes to several other units, for example, by providing multiple nucleic acid analog segments on the same unit. Alternatively, certain nucleic acid analogs may allow for degenerate hybridization schemes such that one nucleic acid analog segment may specifically hybridize to more than one other nucleic acid analog segment, creating a “master key” unit. (See the international application entitled “New Nucleic Acid Base Pairs” submitted herewith, for an example of such segments.) This unique feature increases the flexibility of the instant compositions and methods even further.
Moreover, the instant compositions and methods are compatible with a large variety of samples and are adaptable to a large number of targets, probes, and detectable labels. The present invention is useful in in situ hybridization (ISH), but can be applied to other detection methods as well, such as immunohistochemistry (IHC), immunocytochemistry (ICC), flow cytometry, enzyme immuno-assays (EIA), enzyme linked immuno-assays (ELISA), blotting methods (e.g. Western, Southern, and Northern), labeling inside electrophoresis systems or on surfaces or arrays, and precipitation, among others. Such detection formats, for example, are useful in research as well as in diagnosing diseases or conditions. Further, if multiple targets are detected, such systems may be useful in analyzing expression patterns of genes or levels of proteins within a sample.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
See U.S. Provisional Application No. 60/695,409, and a related co-pending International Application entitled “New Nucleic Acid Base Pairs” for additional information on these non-natural pairing schemes both of which are hereby incorporated by reference herein.